Somatotropin compositions mixed with vitamins

ABSTRACT

The present invention relates to a pharmaceutical compositions which comprises bioactive somatotropin and at least two kinds of lipid-soluble vitamins, and more particularly to a parenterally administered pharmaceutical composition which can solve inconvenience of administering somatotropin and lipid-soluble vitamins respectively and which shows the sustained effect of somatotropin and the synergic effect of somatotropin and lipid-soluble vitamins.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical composition whichcomprises bioactive somatotropin and at least two kinds of lipid-solublevitamins, and more particularly to a parenterally administeredpharmaceutical composition which can solve inconvenience ofadministering somatotropin and lipid-soluble vitamins respectively andwhich shows the sustained effect of somatotropin and the synergic effectof somatotropin and lipid-soluble vitamins.

BACKGROUND OF THE INVENTION

Recently, somatotropin has been produced massively by using geneticengineering techniques. Bovine somatotropin has been commercialized toincrease the productivity of milk and porcine somatotropin has beencommercialized to improve feed conversion ratio and to improve meatquality and so on.

Most of bioactive somatotropin formulations developed until now are thesustained-releasing type that a large amount of somatotropin isadministered and released slowly only to avoid the inconvenience ofdaily administration. For examples, U.S. Pat. No. 5,411,951 and U.S.Pat. No. 5,474,980 disclose the sustained-releasing composition producedby adding a gelling agent such as aluminum monostearate into vegetableoil, and by gelatinizing oil by heating, and by mixing somatotropinhomogeneously. These techniques have been already used to preparesustained-releasing composition of antibiotics (U.S. Pat. No. 2,491,537,U.S. Pat. No. 2,507,193, U.S. Pat. No. 3,016,330), pamoate salts ofoxazepin (U.S. Pat. No. 3,676,557) or relaxin (U.S. Pat. No. 2,964,448),parathyroid stimulating hormone (U.S. Pat. No. 3,869,549), luteinizinghormone releasing factor (U.S. Pat. No. 4,256,737), gonadotropin (U.S.Pat. No. 3,852,422) and insulin (U.S. Pat. No. 2,143,590, U.S. Pat. No.2,174,862, U.S. Pat. No. 2,920,014, U.S. Pat. No. 3,102,077) and thelike.

There are similar techniques for preparing sustained-releasing type byusing oil. For example, EP 211691 discloses that somatotropin is mixedwith wax and oil complex and EP 213851 suggests that sustained-releasingformulation is prepared by mixing somatotropin with oil and glyceriderelease-modifying agent available commercially. And EP 314421 disclosesthat sustained-releasing somatotropin composition is prepared by addingabsorption-controlling material such as calcium stearate and dextran tooil. But this is a formulation that active ingredient has beensubstituted with somatotropin in the known oil-injection formulation.

In addition, the sustained-releasing techniques without using oil havebeen attempted. For example in EP 193917 somatotropin was mixed withwater-soluble polysaccharides such as starch and dextrin to improve thesustained-releasing effect. But this formulation has shorter releasingtime than other formulations mixed with oil and the somatotropin isunstable with water-soluble ingredients.

Another technique which does not use oil to prolong the releasing timehas been described in U.S. Pat. No. 5,520,927. It discloses theformulation of tocopherol acetate and a release-delaying agent. However,in this case tocopherol acetate is used only for delaying the release ofdrug.

Different techniques from the above-mentioned have been attempted forsustained-releasing somatotropin formulations. U.S. Pat. No. 4,861,580discloses sustained-releasing somatotropin formulation is prepared as aliposome type by using lipid-soluble material such as phosphatidylcholine, phosphatidyl ethanolamine and alpha-tocopherol hemisuccinatetris salt. And in U.S. Pat. No. 4,675,189 sustained-releasingsomatotropin formulation was prepared as a microcapsule type by usingbio-compatible polymer. And in U.S. Pat. No. 4,857,506sustained-releasing somatotropin formulation is prepared as a multiplewater-in oil-in water emulsion. But these are inadequate to becommercialized since the processes are so complex and high technologiesare required. Furthermorer the recovery rate to produce the desirablesomatotropin is too low to be commercialized. In addition thesomatotropin formulations prepared by the process are not stable and cannot show the desirable sustained-release effect.

By using quite different methods, solid formulations which areimplantable were prepared for improving the sustained-release of drugs.These techniques have been described in U.S. Pat. No. 4,452,775, U.S.Pat. No. 4,761,289, U.S. Pat. No. 4,765,980, U.S. Pat. No. 4,786,501,U.S. Pat. No. 4,863,736, U.S. Pat. No. 5,035,891, U.S. Pat. No.5,198,422, U.S. Pat. No. 5,228,697, U.S. Pat. No. 5,356,635, U.S. Pat.No. 5,595,752 and EP 246540, 462959, PCT/US92/01877, PCT/US91/08129,PCT/US90/01340, PCT/AU87/00139 and the like. In these techniques solidsomatotropin compositions were prepared and implanted into the animalbody by surgical operation or by using special instruments which areexpensive. The implanting techniques make it possible to release thedesirable amount of bioactive somatotropin during the desirable period.However, the implanting process is too difficult to be performed andanimals also feel uncomfortable due to the foreign substance.

The inventors of the present invention have conducted the intensiveresearch for the sustained-release formulation of somatotropin to solvethe above-mentioned problems. The inventors have found that somatotropinmixed with lipid-soluble vitamins at proper proportions shows theexcellent sustained-release effect and additionally the synergic effectof the active ingredients by administering parenterally.

SUMMARY OF THE INVENTION

The object of the present invention is to provide the pharmaceuticalcompositions, when it is parenterally administered, which comprisessomatotropin and at least two kinds of lipid-soluble vitamins, which hassuch a sustained-release effect that it reduces the pain and the laborcost due to the frequent injections and such a synergic effect of theactive ingredients that it increases the productivity of milk in dairycattle and reproduction efficiency and it decreases the somatic cellcount in the milk, the incidence of mastitis and metabolic diseases ofcattle.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical compositions whichcomprises somatotropin and at least two kinds of lipid-soluble vitamins.

The lipid soluble vitamins which are used in the present inventioncontain vitamin A, vitamin D and vitamin E which are preferably used inpreparing protein drugs since they make protein drugs such assomatotropin stable by preventing protein drugs from binding with water.

In addition to the above-stated advantage of the formulations, thevitamins have its own bioactivities described below.

Particularly, vitamin A promotes the sense of vision especially theadaptability to darkness which is related with rhodopsin and iodopsin ofrod cell and cone cell sensing light on the retina. It can also improveabnormal dryness, denaturalization, keratinization and damage of mucosalmembrane, ophthalmoxerosis and ophthalmomalacia and increase theresistance against various diseases. In addition, it has been reportedto be an essential element in maintaining epidermal tissue and helpingthe growth of bone and teeth and it plays a great role as a growthstimulating hormone.

Vitamin D is an anti-rickets factor. When it is deficient, rickets,osteomalacia, osteopsathyrosis, tetany and the like can be caused. Sincevitamin D is especially important for pregnant or lactating animals orand infant animals, it is necessary to administer by injection when itis deficient by provender.

Vitamin E is related to white muscle diseases. When it is deficient,white muscle diseases are caused, which partly changes the color ofmuscle fiber into gray and makes the muscles atrophied. The muscleatrophy provokes symptoms such as losing flexibility, stiffening andparalyzing of muscle. As the disease progresses, it becomes difficult tobreathe. Resultantly animals having severe symptoms of the muscleatrophy became unable of lactation. Vitamin E is a anti-sterilityvitamin for overcoming the sterility and it also promotes growth rate.And it helps reproducing processes, inhibits the abnormal development ofmuscle and prevents cerebromalacia, irregular activity of muscle,stiffness of muscle, malfunction and tonic spasm.

Hitherto, pharmaceutical compositions have been developed consideringonly the productivity for target animals and the convenience of dairyfarmers. That is, when somatotropin is administered to animals, it isfocused to reduce the dairy farmer's labor and costs of frequent dosageby elongating the releasing time. However, these pharmaceuticalcomposition has been developed by focusing on the view only for thedairy farmers, which may cause unfavorable side effects since the healthconditions of animals are neglected. It is known that the incidence ofmastitis and a major disease of cow depends on the hygienic condition ofsurrounding environment, especially milking condition. But recently ithas been reported that the incidence of mastitis depends on the totalmilk production of each dairy cow, that is, the more the productivity ofmilk increases, the more the incidence of mastitis increases. Thereforewhen somatotropin is used to increase the milk productivity, the propermanagement should be followed. To prevent mastitis hygienic conditionshould be necessary and the proper management program for the superiorcow should be taken

There are many factors to cause the mastitis as mentioned in the above,but it is more important to increase cow's resistance against bacteria.To increase the resistance against bacteria, proper drugs such asvitamins must be provided, in addition to the above-stated management.Practically, when vitamins are deficient the mucosal epithelial cells ofpapillary duct and papillary sinus are keratinized, which induces theinfection and multiplication of bacteria. And the multiplied bacteriainvade papillary sinus, which results in the severe inflammation. Inaddition the multiplied bacteria inhibit the synthesis of keratin,protecting material within papillary duct, and immunoglobulin. Thiscauses the serious mastitis.

There are several methods for examining the mastitis, but the generalmethod is to count the number of somatic cells in milk. The criteria fordiagnosis is the number of somatic cells per 1 ml of milk. The lower thenumber the better the milk. And it can be passed that it is producedfrom cow without the clinical mastitis.

Therefore, in the present invention pharmaceutical compositions whichcomprises somatotropin and at least two kinds of lipid-soluble vitaminswhich do not affect the stability of somatotropin has been developed toenhance the productivity of milk and the health of animal by minimizingthe side effects due to the increase of milk productivity.

Among lipid-soluble vitamins, vitamin A and vitamin D may show sideeffects when excessive amounts are administered. Thus the process forpreparing the formulations should be made with extra care. Most ofsomatotropin formulations used recently have been manufactured assustained releasing types which are administered every 2 weeks. Thepresent invention also provides the formulation of sustained-releasingtype having 2 weeks period. And the amount of vitamin A and vitamin D inthe formulation has been controlled properly for preventing side effectsof excessive administration and maximizing the synergic effect of thesomatotropin and vitamins.

The somatotropin of various animals can be used in the presentinvention, but that of cow, pig or the like is preferred. And thenatural somatotropin purified highly from pituitary gland of animals andsomatotropin produced artificially by recombinant DNA technology alsocan be used.

In the pharmaceutical composition of the present invention, thesomatotropin is contained in the range of 10-40 weight%, vitamin A iscontained in the range of 500,000-5,000,000 units per 1 g ofsomatotropin. And vitamin D is contained in the range of 100,000-700,000units per 1 g of somatotropin and vitamin E is contained in the range of500-4,000 units per 1 g of somatotropin.

The pharmaceutical composition of somatotropin and lipid-solublevitamins is prepared by adding lipid-soluble vitamins to a powderedsomatotropin and homogenizing them. The powder-type somatotropin isprepared by lyophilizing only the somatotropin bulk solution, or bylyophilizing microparticles prepared by mixing delaying agent such aslecithin and the somatotropin bulk solution, or by lyophilizing themixture of stabilizing agent such as sucrose, mannitol, trehalose andthe somatotropin solution.

When the lyophilized somatotropin powder is used, water content andparticle size should be considered. The water content concerned with thestability of the somatotropin should be below 3%. And the particle sizeconcerned with the injection and the layer-separation after long storageshould be below 10 μm in diameter. Therefore the lyophilizedsomatotropin should be ground to reduce the size when the size of theparticle is above the range. At that time, the process can be performedwith ball mill, air mill or the like, not changing the quality of theparticle.

The pharmaceutical composition in the present invention is prepared byadding lipid-soluble vitamins to the powdered somatotropin prepared asthe above-mentioned method in the proper ratio.

Preferred embodiments of the invention are illustrated as shown in thefollowing examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention

EXAMPLES Example 1

500 ml of bovine somatotropin (LG Chemical Ltd. Biotech. Institute)solution (60.5 mg/ml) was mixed with 10.08 g of lecithin by using ahomogenizer for 30 min. and was grounded in a microfluidizer to makeparticles of less than 200 nm size of diameter. The suspension wasfiltrated by using a filter of 0.22 um pore size for sterilization andautoclaved, then dried in a freeze dryer. At that time the lyophilizingprocess was performed by using bottles in vacuum below 100 milli torr,at −70° C. and for about 48 hours. Bovine somatotropin-lecithinlyophilized as a powder form has 1.4% of water content which is measuredin a boiling counter.

And 267 g of vitamin A palmitate (1 g =1,000,000 unit, viscous solution,BASF) and 1 g of vitamin D₃ (1 mg=40,000 unit, powder, Sigma) werequantified and mixed by using a magnetic bar.

Then 6.67 g of bovine somatotropin-lecithin (5 g bovine somatotropin,1.67 g lecithin) as lyophilized powder was evenly mixed with 6.93 g ofvitamin E acetate (1 mg=1 unit, viscous solution, ROCHE) and 7.53 g ofthe above mixture of vitamin A and vitamin D₃ in a homogenizer. Then themixed composition was put in a vacuum chamber for 6 hours to remove airbubbles.

Example 2

The pharmaceutical composition was prepared by performing the sameprocess of Example 1 with different component ratios. 6.67 g of bovinesomatotropdn-lecithin as lyophilized powder was evenly mixed with 9.44 gof vitamin E acetate and 5.02 g of the mixture of vitamin A and vitaminD₃ in a homogenizer.

Example 3

The pharmaceutical composition was prepared by performing the sameprocess of Example 1. 6.67 g of bovine somatotropin-lecithin aslyophilized powder was evenly mixed with 3.16 g of vitamin E acetate and11.30 g of the mixture of vitamin A and vitamin D.

Example 4

The pharmaceutical composition was prepared by performing the sameprocess of Example 1. 6.67 g of bovine somatotropin-lecithin aslyophilized powder was evenly mixed with 11.95 g of vitamin E acetateand 2.51 g of the mixture of vitamin A and vitamin D₃.

Example 5

The pharmaceutical composition was prepared by performing the sameprocedure of Example 1. 3.34 g of bovine somatotropin-lecithin aslyophilized powder was evenly mixed with 6.93 g of vitamin E acetate and7.53 g of the mixture of vitamin A and vitamin D₃.

Comparative Example 1

The composition was prepared by performing the same process ofExample 1. 6.67 g of bovine somatotropin-lecithin as lyophilized powderwas mixed with 14.46 g of vitamin E acetate.

Comparative Example 2

The composition was prepared by performing the same process ofExample 1. Precisely 3.34 g of cow somatotropin-lecithin as lyophilizedpowder was mixed with 14.46 g of vitamin E acetate.

Example 6

The animal test was performed by using the somatotropin compositionswhich were prepared in Example 1, 2, 3, 4 and Comparative Example 1 andby utilizing the porcine somatotropin powder without lecithin instead ofthe above bovine somatotropin-lecithin powder. As experimental animals,rats having genetic dwarfism were utilized.

Female, 8-week old dwarf rats weighed about 100 g were used. Theinterval between light and dark was 12 hours and water and feed wereprovided freely. 4 rats in a cage were used for examining the effects ofone composition. The rats were separated randomly according to theaverage weight during 3 days and the standard error measured beforeadministration. The average weight during the 3 days was considered as astandard weight. 0.04 ml of each composition (corresponding to 10 mg ofporcine somatotropin) was subcutaneously injected in the abdominal partand then the weights were measured at the determined time every day for7 consecutive days. As a comparative group, 4 dwarf rats without theinjection were examined and the weights were measured during the aboveperiod by the same process. The cumulative mean weight gain wasindicated in table 1 which was measured at the animal group respectivelyand cumulatively.

TABLE 1 Comp. Day Example 1 Example 2 Example 3 Example 4 ExampleControl 1  9.95 ± 1.77 10.18 ± 0.08  9.34 ± 0.29 10.66 ± 0.90  9.66 ±0.69 0.88 ± 1.61 2 17.35 ± 1.85 17.93 ± 0.08 17.74 ± 0.86 17.71 ± 1.4018.61 ± 0.87 0.68 ± 1.08 103  24.15 ± 1.01 25.16 ± 0.90 24.37 ± 0.7723.76 ± 1.81 24.30 ± 0.98 2.62 ± 1.05 4 30.95 ± 0.54 32.38 ± 1.01 31.27± 0.68 29.81 ± 2.29 29.98 ± 1.09 3.55 ± 1.03 5 37.03 ± 1.44 38.33 ± 1.3936.42 ± 0.17 33.98 ± 4.11 34.53 ± 0.98 3.70 ± 0.63 6  4.40 ± 2.04 44.01± 1.25  4.22 ± 0.60 38.48 ± 3.98 40.46 ± 1.57 6.45 ± 0.97 7 40.58 ± 1.7341.23 ± 1.43 40.47 ± 1.93 35.13 ± 2.90 38.48 ± 1.42 5.08 ± 0.64 (mean +standard error: g)

As shown in the table 1, in the group administered the pharmaceuticalcomposition of the present invention, the weights of animals increasesmore than about 30 g than that of the control group at day 7.

Example 7

Experiment has been conducted to the cow which are main target animalsof the present pharmaceutical composition by using the pharmaceuticalcompositions prepared in Example 1, 2, 3 and Comparative Example 1. Asexperimental cow, Holstein species having the parity more than twice andthe lactation day between 65-186 days were selected. Each cow wasdiagnosed to be healthy before the experiment and particularly mastitiswas examined by culturing bacteria respectively. In order to make themilk yield of each group evenly at the starting point of the experiment,the mean dairy milk yield of each cow was calculated and then 45 cowsare divided into 5 groups, in order for each group to have similar milkyield. At the starting point, the milk yield, lactation day and paritywere depicted in table 2. In the each group, the mean yield of milk, theparity and the lactation day were in the narrow range of27.4±2.9-27.8±3.1 kg, 2-7 parity and 65-186 days respectively.

TABLE 2 min. max. standard index value value mean error control milk15.3 38.2 27.8 3.1 yield (kg/day) parity 2 6 3.4 0.5 lactation 85 185137.7 11.4 day Example 1 milk 21.1 38.1 27.8 2.1 yield parity 2 6 3.40.4 lactation 65 186 136.3 12.8 day Example 2 milk 16.1 40.3 27.7 2.9yield parity 2 6 3.8 0.5 lactation 88 186 138.4 12.1 day Example 3 milk20.0 34.0 27.8 1.6 yield parity 2 5 3.8 0.3 lactation 66 174 140.4 11.3day Comp. 16.9 37.3 27.4 2.9 Example 1 2 7 3.9 0.6 79 177 127.7 13.1

Considering the above parity, lactation day and the like, 45 cow wereseparated into 5 groups containing 9 heads per group, particularlynegative control group where nothing had been administered, group 1administered with 500 mg (bovine somatotropin 250 mg/ml) of thepharmaceutical composition prepared in Example 1 and mixed with vitaminA, D and E (A: 750,000 unit; D: 112,500 unit; E: 693 unit), group 2administered with 500 mg (bovine somatotropin 250 mg/ml) of thepharmaceutical composition prepared in Example 2 and mixed with vitaminA, D and E (A: 500,000 unit; D: 75,000 unit; E: 944 unit), group 3administered with 500 mg (bovine somatotropin 250 mg/ml) of thepharmaceutical composition prepared in Example 3 and mixed with vitaminA, D and E (A: 1,125,000 unit; D: 168,750 unit; E: 316 unit) and theother comparative group 1 administered with 500 mg (bovine somatotropin250 mg/ml) of the pharmaceutical composition prepared in ComparativeExample 1. These compositions were injected subcutaneously through rightor left ischiorectal fossa alternatively. Before the injection, theinjection sites were sterilized completely by using 70% EtOH cottoncontaining iodine and after the injection, efficient massage wasperformed for about 30 seconds in order to spread the drug evenly.During the experimental period, the amount of concentrated dietsprovided was controlled according to that of milk produced and cornsilage and alfalfy hay was provided freely. The other condition werecontrolled by the regulations of the general experimental farm. During 1week before injection, milk yield produced daily were measured in orderto get the standard value of milk yield and compared with the daily milkyield produced after the injection. And the contents of milk fat, milkprotein, milk lactose and milk solid not fat) in the milk collected fromeach cow were analyzed by using the automatic milk analyzer (Milk-Scan133B, Foss Electric, Denmark). The above milk was collected in themorning and afternoon respectively and was mixed according to the ratioof the productivity at the 1 week before injection and every 4 weekafter the injection. During the total period, each cow's milk was alsocollected sterilely every 4 week, inoculated into sheep blood agar mediaand cultured for identifying the species of bacteria in order todiagnose mastitis, And the results were analyzed. And strip cup test andsterilization of udder were also performed before and after everymilking. As an index of mastitis, somatic cell count was measured in theeach milk collected before and after the injection by using Foss-O-maticSomatic Cell Count (Foss Electric, Denmark). The milking procedure wasadopted from regulations of the general experimental farm and theoperating conditions of the milking machines was checked every monthbefore the experiment. During the overall period body condition scorewas measured every 4 week. The results were shown in table 3. The table3 indicated milk yield, increase ratio of milk yield to that ofcomparative group, component variation of milk, body condition score andindex of mastitis (somatic cell count).

TABLE 3 Comp. control Example 1 Example 2 Example 3 Example 1 milk 21.426.0 27.2 27.6 26.2 yield (kg/day) increase — 21.5 27.1 29.0 22.4 rate(%) of milk yield to compara- tive group milk fat 3.44 3.45 3.59 3.463.28 (%) milk 3.41 3.46 3.45 3.44 3.37 protein (%) milk 4.88 4.85 4.784.93 4.69 lactose (%) milk 9.07 9.01 9.08 9.13 8.81 solid not fat (%)somatic 557 348 507 291 463 cell count (10³/ml) body 3.0 2.8 2.8 2.7 2.7condi- tion score

As shown above, the pharmaceutical compositions of the present inventioncomprising somatotropin and at least 2 kinds of lipid-soluble vitaminsenhance the growth of animal and the milk yield of cow and improved thehealth outstandingly by preventing mastitis, a major problem.

Considering the general practice of administering somatotropin andvitamins by separate injections, the pharmaceutical composition of thepresent invention has many advantages of reducing costs, labor and painsof target animals due to single injection.

As described above, the present invention relates to the pharmaceuticalcompositions comprising somatotropin and at least two kinds oflipid-soluble vitamins, which can be administered parenterally such asby injection. The compositions has sustained and synergic effect byadministering somatotropin and vitamins at once, thus increase the milkyield and the reproduction performance and reduce the incidence ofmetabolic diseases such as mastitis and reduce the frequency ofadministration. Therefore they also effectively reduce costs, labor andpains of target animals by low frequency of administration.

What is claimed is:
 1. A method for increasing milk production anddecreasing incidence of mastitis of dairy cattle, which comprises thesteps of: (a) providing dairy cattle; and (b) administering to saiddairy cattle a pharmaceutical composition comprising somatotropin, adelaying agent and at least two kinds of lipid-soluble vitamins whichare selected from the group consisting of vitamin A in the range of500,000-5,000,000 units per 1 g of somatotropin, vitamin D in the rangeof 100,000-700,000 units per 1 g of somatotropin, and vitamin E in therange of 500-4,000 units per 1 g of somatotropin.
 2. The methodaccording to claim 1 wherein the somatotropin is bovine somatotropin orporcine somatotropin.
 3. The method according to claim 1 wherein thesomatotropin is bovine somatotropin produced by recombinant DNAtechnology.
 4. The method according to claim 1 wherein the somatotropinis contained in the range of 10-40 wt %.
 5. The method according toclaim 1, wherein the delaying agent is lecithin.